Project Summary/Abstract Heart muscle cells (cardiomyocytes) beat over 100,000 times per day. Successful heart contraction requires that cardiomyocytes be physically coupled. This is accomplished through the intercalated disc (ICD): a specialized adhesive structure joining cardiomyocytes end-to-end to form a functional syncytium. The ICD contains protein complexes responsible for mechanical strength and protein complexes responsible for electrical conductance. The adherens junction (AJ) is a protein complex responsible for mechanical strength and integrates the actin cytoskeletons of neighboring cells. Myofibrils, organized actin-myosin structures, are the contractile machinery of the heart and produce the force necessary for individual cardiomyocytes to beat. Myofibrils terminate in the ICD, and linking myofibrils between cardiomyocytes allows for mechanical continuity across cells. However, how these myofibrils are coupled between cells is largely unknown. Vinculin is a key protein mediating cell-cell and cell-extracellular matrix (ECM) contacts, serving to link both complexes to the actin cytoskeleton. Vinculin is recruited to AJs in a tension-dependent mechanism and is enriched at the AJ-myofibril interface in cardiomyocytes. Expression of vinculin at cell-cell contacts increases in aging heart muscle and is thought to be cardio-protective, and disorganization of vinculin is seen in failing heart muscle. Yet the role of vinculin at the ICD is poorly understood. In this proposal, it is hypothesized that vinculin anchors myofibrils to AJs and mediates crosstalk between cell-cell and cell-ECM contacts. In Aim 1, the linkage between myofibrils and AJs will be investigated by defining the myofibril-vinculin-AJ interface and determining the role of vinculin and its AJ binding partner, ?E-catenin in promoting AJ stability. In Aim 2, the crosstalk between AJs and cell-ECM contacts will be investigated by altering the ECM environment and determine its influence on vinculin localization, dynamics, and tension at cell-ECM contacts and AJs. The work proposed will further the understanding of a fundamental aspect of cardiac biology: how individual cardiomyocytes are coupled to create a functional syncytium. This research will elucidate vinculin function in the heart, shedding light on to its cardio-protective role.